Abstract

We conducted a quantitative analysis of the extent of de
novo methylation of four CpG islands in human urinary
transitional cell carcinomas of different stages and grades to
determine how frequently these CpG islands became methylated in
transition cell carcinomas during progression. The CpG islands included
exon 5 of PAX6, exon 2 of p16, the 5′ end
of the deleted in bladder cancer gene, and the 5′ end of transmembrane
protein containing epidermal growth factor and follistatin domains.
These sequences were not methylated in normal urothelial tissues;
however, 48 of the 54 tumors examined (89%) showed methylation levels
in excess of 20% for at least one of the markers. The number of
markers concurrently methylated in individual tumors increased with the
stage of the tumor, with several of the more aggressive invasive
cancers showing hypermethylation of all four markers compared with
the less aggressive invasive cancers. However, considerable methylation
defects were present in superficial, preinvasive, papillary tumors.
These data demonstrate that 89% of bladder cancers have increased
methylation of CpG islands relative to their normal counterparts and
suggest the occurrence of a hypermethylator phenotype in which
multiple independent CpG islands become concurrently methylated in
individual tumors in a process associated with tumor progression.

INTRODUCTION

Alterations in the methylation patterns of the genome are among
the most common genetic changes observed in human cancers
(1, 2)
, though the true extent of these changes is not yet fully
known. Analysis of the methylation patterns of candidate genes,
particularly tumor suppressor genes such as p16,
RB, and VHL, has convincingly demonstrated that
the promoters of these genes become altered and contribute to
carcinogenesis
(3,
4,
5,
6,
7)
. However, the analysis of candidate
genes does not give a comprehensive picture of the totality of changes
occurring in a given tumor class because these analyses are, by their
nature, highly focused. Also, although many studies have demonstrated
methylation changes in cancers, the methods used are not always
quantitative, and therefore, it is difficult to appreciate the full
extent of the changes from the semiquantitative data obtained.

Recently, several investigators have developed genome-scanning
techniques sensitive to DNA methylation to gain an appreciation of the
genome-wide changes occurring within various cancers. These
approaches, which include methylation-sensitive arbitrarily
primed PCR
(8,
9,
10)
, methylated CpG islands amplification
(11)
, and
restriction landmark genomic scanning
(12)
among others, have shown
that methylation changes are widespread, but it has not always been
possible to quantitate the extent of changes from these screening
techniques. Our earlier analyses by genome scanning of bladder,
prostate, and colon cancers suggested the possible occurrence of a
hypermethylator phenotype
(13)
. Toyota et
al.(14)
have recently described a
hypermethylator phenotype in colorectal cancers that they called
CIMP (CpG island methylator phenotype). Therefore, we have extended our
earlier semiquantitative studies to use a quantitative technique
developed in this laboratory for the assessment of methylation
(15)
to measure the levels of inappropriate methylation of
CpG islands, which have been observed to become methylated in various
cancers.

We focused our studies on human bladder cancer, the 5th most
common cancer in the United States.
TCCs
4
are clinically managed according to their stage and grade. Currently,
superficial (preinvasive) papillary bladder cancers and
non-muscle-invasive (minimally invasive) bladder cancers are treated
conservatively, whereas muscle-invasive bladder cancers are treated
more aggressively. Furthermore, it has been shown previously that
superficial (preinvasive) bladder tumors are genetically distinct from
invasive bladder tumors with different aberrant molecular profiles
(16)
. We have conducted a quantitative methylation
analysis of four loci in bladder tumors of different grades and stages
to determine whether methylation changes occur early in the
carcinogenic process and/or become reinforced during tumor progression.

Quantitation of Methylation by Ms-SNuPE Analysis.

Methylation was quantitated using a Ms-SNuPE assay, described by
Gonzalgo and Jones in 1997
(15)
. Briefly, genomic DNA was
treated with sodium bisulfite to convert unmethylated cytosines to
uracil (which then converts to thymine after PCR) and leave
5-methylcytosines unchanged. Amplification of the desired target
sequence was performed using PCR primers specific for
bisulfite-converted DNA, and the PCR products then were isolated and
used as a template for methylation analysis at the two or three CpG
sites by two or three specific Ms-SNuPE primers (Table 1)
⇓
. The final PCR products were then resolved on a 15% polyacrylamide
gel and a PhosphoImager (Molecular Dynamics, Sunnyvale, CA) was
used to quantitate the percent methylation averaged from the three CpG
sites.

Statistical Analysis.

Two-sided Ps were generated for each tumor group and
compared with normal controls using the Wilcoxon rank sum test. The
significance of an increasing methylation trend between minimally
invasive tumors and the muscle-invasive tumors was determined using a
Mantel-Haenszel χ2 test
(18)
.

RESULTS

We analyzed four CpG islands known to undergo de
novo methylation during bladder carcinogenesis. The exon 5
region of PAX6 was selected because this small intronic CpG
island was found to be methylated frequently in a genomic screen
(13)
, although its methylation is not associated with
down-regulation of the gene
(19)
. The exon 2 region of
p16 was selected because this region is frequently
methylated in bladder cancer cell lines and tumors
(3)
,
and the 5′ end of the DBC gene was studied as a potential
candidate suppressor gene in bladder cancer known to be methylated in
cell lines
(20)
. The fourth region studied was located in
the 5′ end of a previously unknown gene, TPEF, which was
also isolated from the arbitrarily primed-PCR screen previously
conducted
(13, 21)
. Both the latter two regions are
associated with the 5′ region of their genes.

The methylation levels of these regions were analyzed by the
quantitative Ms-SNuPE analysis
(15)
, which was
standardized in each case to ensure the linearity of the PCR
amplification of the region after bisulfite treatment. Typical Ms-SNuPE
results for the four regions investigated are shown in Fig. 1
⇓
, in which extensive methylation of three of the four CpG islands is
shown by the increased intensities of bands in the C lanes
of the analysis. Therefore, this method gives a quantitative assessment
of the methylation status of specific CpG sites within each CpG island
region.

Ms-SNuPE analysis (patient 487). Quantitative methylation
analysis of two or three CpG sites in PAX6 exon 5,
p16 exon 2, 5′ end of DBC, and 5′ end of
TPEF using the Ms-SNuPE technique. The percent
methylation represents the average of two or three sites using the
following equation on PhosphoImager quantitation:
(methylated)C/[(methylated)C + (unmethylated)T] × 100.

Figs. 2
⇓
and 3
⇓
summarize the data from the methylation analysis of the 58 tissue
specimens collected. All of the loci for each of the four normal
tissues were found to be methylated to a level less than 12%,
averaging 5% overall (Fig. 2)
⇓
. This low amount of methylation was as
expected because CpG islands on autosomal genes are generally
unmethylated in normal tissues
(22)
. We chose 20% as the
cutoff for significant methylation to avoid false positive
interpretations from our data.

Proportion of patients with zero to four CpG islands
methylated over 20% in each group of the tissues examined. A
statistically significant trend (P = 0.047) is shown with increasing methylation in progressively invasive
tumors.

Each of the tumor tissue groups were found to be significantly
more methylated than the normal controls (P = 0.002–0.0001; Fig. 2
⇓
). Increased methylation of some of the loci was
seen in the minimally invasive tumors (pT1) with three of the five
(60%) showing methylation levels greater than 20% of at least one of
the markers examined. In contrast, the muscle-invasive tumors obtained
from patients without lymphatic metastases (>pT2N0) showed methylation
levels greater than 20% of at least one of the markers in 12 of the 13
tumors (92%) examined. Furthermore, the degree of CpG island
methylation was also substantial in the muscle-invasive tumors with
associated metastatic lymph nodes (>pT2N+), with 15 of the 16 tumors
(94%) in this group showing methylation of greater than 20% of at
least one of the markers. Interestingly, many of the tumors had
multiple methylation changes with increased methylation greater than
20% seen at all four loci in a considerable proportion of the tumors
(Fig. 3)
⇓
. These data clearly show that the degree of aberrant
methylation in bladder cancer increases substantially as the tumors
progress from minimally invasive (pT1) to muscle-invasive (>pT2N0) to
muscle-invasive with metastatic lymph nodes (>pT2N+);
P = 0.047. The levels of methylation of the
four CpG islands examined in the primary tumors of patients 79, 11, 13,
19, 24, 15, and 23 were generally conserved or increased in the seven
metastatic lymph nodes obtained from the same patients (Fig. 2)
⇓
. This
suggests that further increases in cytosine methylation may accompany
tumor metastasis.

Figs. 2
⇓
and 3
⇓
also show that substantial methylation changes were
already present in the preinvasive (papillary) tumors with 8 of the 9
(88%) of these tumors showing methylation greater than 20% of at
least one marker. Interestingly, the methylation changes were mainly
confined to the PAX6 exon 5 and p16 exon 2
regions, and little increased methylation was seen in the two islands
located in the 5′ regions of DBC or TPEF.

Most of the tumors examined in this study were grades 3 and 4 (Fig. 2)
⇓
,
and there was no statistically significant relationship between grade
and methylation level. Although many tumors had increased methylation
of both the p16 and PAX6 markers, the methylation
levels in each marker were not highly correlated with each other
(correlation coefficient = 0.37).

DISCUSSION

The results of this quantitative analysis of the methylation
levels of four independent CpG islands in human bladder cancer
underscore the commonality with which these changes are observed in
TCC. The four CpG islands selected for study were chosen because of
their propensities to undergo de novo methylation as
observed in earlier studies. The regions selected included two
transcribed regions where methylation is not considered to be
involved in gene silencing and two regions located in the 5′ region of
genes where they might be more likely associated with transcriptional
silencing. The data in this study showed that considerable methylation
changes are present in both preinvasive (papillary) and invasive TCCs
of the bladder, with a statistically significant trend toward
increasing methylation in the more aggressive tumors.

Furthermore, this study showed that CpG islands differ in their
propensities to become de novo methylated. Thus, the two CpG
islands in the transcribed regions of genes were hypermethylated in
almost every tumor specimen examined, whereas methylation of the 5′
regions of the genes was considerably less frequent. Another
observation is that although multiple CpG islands became methylated in
advanced tumors, each island appeared to behave independently of the
others and there was no direct correlation present between methylation
of the p16 exon 2 and PAX6 region, for example.
Thus, although TCCs have frequent methylation of CpG islands suggesting
the presence of a hypermethylator phenotype, the extent of methylation
of each island was apparently independently acquired in each tumor. It
was also interesting that the noninvasive papillary tumors had more
frequent methylation changes than the minimally invasive tumors, which
are more likely to progress to invasive disease. Because we have
previously shown that these two types of tumors harbor distinct genetic
abnormalities
(16)
, these epigenetic changes may also
reflect the different pathologies and outcomes of these two tumor
types.

Previous studies related to the importance of DNA methylation in
human cancer have focused on regions of the genome that might have
functional significance in the extinction of gene activity. Using a
quantitative scan of CpG islands located in different regions of the
transcriptional unit suggests a hierarchy of changes that might explain
how promoter methylation is achieved during cancer progression. Work
with transgenic animals has shown that the presence of a functional
promoter blocks de novo methylation during embryonic
development
(23, 24)
. Thus, the occupation of a CpG island
in a promoter by a transcription initiation complex may make the
promoter more resistant to de novo methylation than a
nonprotected transcribed region
(25)
. Our data suggest
that CpG island methylation occurs more frequently in regions of DNA
that are downstream of the transcriptional start site, making these
changes one of the most common genomic alterations in human bladder
cancer.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

↵1 Supported by USPHS Grant IR01 CA83867 from the
National Cancer Institute and by the American Foundation for Urologic
Disease.